Blood count reference intervals for the Brazilian adult population: National Health Survey

ABSTRACT Objective: To estimate the reference intervals (RIs) of complete blood count parameters in the Brazilian adult population. Methods: Cross-sectional study, with data from the National Health Survey (Pesquisa Nacional de Saúde – PNS), between 2014–2015. The final sample consisted of 2,803 adults. To establish the RIs, exclusion criteria were applied, outliers were removed and partitions were made by gender, age, and race/skin color. The non-parametric method was adopted. Differences were assessed using the Mann Whitney and Kruskal Wallis tests (p≤0.05). Results: There were statistically significant differences for the following hematological parameters based on gender, red blood cells, hemoglobin, hematocrit, MCH, MCHC, eosinophils and absolute monocytes, neutrophils and platelets (p≤0.05). When analyzed by age, the RIs were statistically different in females for hematocrit, MCV, white blood cells and RDW and in males for red blood cells, white blood cells, eosinophils, mean platelet volume, MCV, RDW, and MCH (p≤0.05). For race/color, there were differences in the RIs for parameters of hemoglobin, MCH, MCHC, white blood cells and mean platelet volume, neutrophils and absolute eosinophils (p≤0.05). Conclusion: The differences found in the RIs of some in blood count parameters in Brazilian adults reaffirm the importance of having their own laboratory reference standards. The results can support a more accurate interpretation of tests, adequate identification and disease prevention in Brazil.


INTRODUCTION
Blood count reference intervals (RIs) (red and white series) are important information in clinical practice 1 for screening blood donors, assessing overall health, establishing effective diagnosis 2 , and managing and treating diseases [1][2][3] .
Reliable RIs direct the identification of diseases of important magnitude, such as anemia, infections and neoplasms, and contribute to control and prevention 4 . Anemia represents a global health problem; in 2019, it corresponded to 1.8 billion prevalent cases in the world 5 , and in Brazil, according to data from the National Health Survey (Pesquisa Nacional de Saúde -PNS), between 2014 and 2015, there was a prevalence of 9.9% in adults and the aged 6 . In 2019, cancer was the second cause of death worldwide (10,079,637), and in Brazil it corresponded to 266,034 deaths. Respiratory tract infections represented the fourth cause of death worldwide (2,493,199), being the third cause in Brazil (88,640) 7 .
However, establishing RIs is a challenge due to the requirement of methodological rigor with the need for a representative sample of the population; care in collection, transport, biochemical and statistical analysis 1,8 . Thus, determining RIs is not a reality in all countries, being restricted to those that conduct population studies 9 .
Furthermore, RIs are influenced by factors such as race, ethnicity, body mass index (BMI) 10 , circadian rhythm, diet, pregnancy, menstrual cycle 11,12 , menopause 11 , physical activity, stress, smoking and use of medications, alcoholic beverages or caffeine 12 . Therefore, it is recommended to determine the RIs for the population in which they will be applied 10,13 , as they reflect the real health conditions 11 .
Even though the importance of having an RIs belonging to the population is recognized, international RIs are adopted in Brazil 1,12 . So far, there is only one study, in which reference values for blood counts in Brazilian adults were calculated using PNS data, using the parametric method 1 .
The population has influences on the RIs values, therefore, advancing in analytical calculation methods can minimize this effect 2 . The application of a single approach to RIs calculation can lead to inaccuracy, and it is recommended to test other methodologies 13 . Thus, it is important to carry out studies using the same PNS database, in which different methods of RIs determination are adopted.
This study analyzed, for the first time, the blood count RIs of Brazilian adults with laboratory data from the PNS using the non-parametric method, and according to the recommendations of Guideline C28-A3 14 , a reference widely adopted by laboratories 13 . In addition, it advanced by expanding the exclusion criteria, including hemogram parameters according to race/color and in the analyses used for partitioning in relation to the study previously carried out in Brazil 1 .
Thus, the present study aimed to estimate the blood count RIs in the Brazilian adult population.

Study design
Cross-sectional study, with PNS data, between 2014 and 2015.

Context and data source
The PNS is a household-based survey carried out by the Brazilian Institute of Geography and Statistics (Instituto Brasileiro de Geografia e Estatística -IBGE) 15,16 . In the 2013 PNS, 60,202 adults were interviewed. The collection of tests was planned in a subsample of 25% of the census sectors of the research, and carried out between 2014 and 2015, in 8,952 adults 16 . Blood counts were collected at any time of the day in tubes filled with ethylenediaminetetraacetic acid (EDTA). The blood samples were sent to the reference laboratories that ensured the quality control of the Ministry of Health. The samples were examined by an automatic cell analyzer 16 .
Due to the complex sampling design of the PNS and the unequal selection probabilities, weights were calculated by post-stratification procedures. Sampling weights were adopted in all analyses 16 . Further details on the PNS sampling plan, and the procedures for collecting, sending and storing samples are available in other publications 15,16 . The database used is available at: https://www. pns.icict.fiocruz.br/.

Determination of blood count reference intervals
In order to reduce the factors that can influence RIs 11 , and aiming to reach a healthy population, exclusion criteria based on the literature 1,8,14 were applied and the criteria adopted in the national study were expanded 1 . The exclusion criteria used, their references and cutoff points 17-21 are in Supplementary Material 1.
The sample was partitioned according to gender, age and race/skin color, using statistical tests 11,14 and considering the biological conditions that influence RIs 11 .
RIs were estimated considering 95% of healthy individuals 9 , linked to the 2.5 and 97.5 14 percentiles. Samples of over 120 individuals were used in partitioning by gender and age 14 .

Participants
Participants were adults aged 18 years old and older. The PNS database, composed of 8,952 individuals, was used. After exclusion procedures and removal of outliers, the final sample consisted of 2,803 participants.

Variables
The variables included were: sociodemographic and hemogram parameters (red and white series). The complete description of the variables can be found in Supplementary Material 2.

Statistical analyses
Medians were calculated for reference limits. The lower limit (LL) was linked to the 2.5 th percentile and the upper limit (UL) to the 97.5 th percentile of the reference population distribution, according to gender, age, and race/color. RIs were estimated using the non-parametric method, which organizes the observations made by size and classifies them considering the lowest r=1 to the highest r=n. LL corresponded to r=0.025 (n+1) and the UL, the ranking position r=0.975 (n+1) 14 .
Data normality was evaluated using the Shapiro Wilk test and differences were assessed using the Mann Withney or Kruskal Wallis tests, with Dun's post-test with Bonferroni correction, with a significance level of 5%.
Analyses were performed using the Data Analysis and Statistical Software (Stata), version 14, and the Software Package for Social Science (SPSS), version 25.0, using the survey module, which considers post-stratification weights.

Ethical aspects
The PNS was approved by the National Research Ethics Committee of the National Health Council (Opinion 328.159). Adult participation was voluntary, and confidentiality of information was guaranteed 15 .
In men, the RIs of red blood cells (millions/mm 3 ) were higher between 18 and 39 years of age (4.4-5.8; median 5.1), compared with the age groups from 40 to 59 years In women, there were differences for LL and UL, and the median was lower for hematocrit (%) between 18  In women, there were differences for RIs of white blood cells (mm 3 ); the median and LL were more prominent between 18 and 39 years of age (2,600-10,000; median 6,300) and 40 to 59 years (2,800-9,800; median 5,800) than at 60 years old and older (2,000-9,800; median 5,500) (p≤0.05) (Supplementary Material 6).

DISCUSSION
In this study, blood count RIs of Brazilian adults was estimated by PNS tests, using the non-parametric method. Statistically significant differences were observed for some components of the red and white series blood count when analyzed according to gender, age, and race/color. RIs were calculated using a methodology that had not yet been tested, following recommendations in the literature, in order to obtain increasingly accurate and reliable values 13 . Therefore, it differed from the only existing national study in which reference values were calculated for Brazilian adults, by using a non-parametric approach and also by the tests applied for sample stratification, application of the Tukey method to remove outliers and the expansion of the exclusion criteria of the study by Rosenfeld et al. 1 .
The non-parametric methodology adopted here is in line with studies carried out in Ghana 3 , Canada 8 , India 9 , Kenya 10 , and Korea 22 . This method is recommended due to many analytes not having a normal distribution, and because it is simpler, depending only on the classifications of the reference data arranged in increasing order of size 14 . The literature describes that the results found in parametric and non-parametric methods are usually similar 14 . As can be seen from the median values found in this study according to gender and the mean values identified in the national study for red and white blood cells were 5.0 million/mm 3 and 6,142 mm 3 in men and 4.5 million/mm 3 and 6,426 mm 3 in women, respectively 1 .
For selection of healthy individuals, exclusions were defined according to Guideline C28-A3 14 and based on studies 1,[8][9][10]23 . The procedures adopted here for removing outliers were used in studies in Canada 8 and Oman 24 . Tukey's method was used because it is more useful and indicated in the presence of more than one outlier 13 , which occurred in this study, and visual inspection because it is considered effective 13 .
Considering partitioning as a power tool for diagnosing RIs 7 , this investigation used statistical tests to verify its need, as well as in other studies [22][23][24][25] . The partitioning adopted is set out in Guideline C28-A3 15 , and the physiological changes of adulthood were also considered 11 . When calculating RIs, the physiological aspects are as important as the statistical ones 11,14 , as RIs differ in children, adults, the aged, men and women; in puberty, pregnancy and menopause, due to organic changes throughout life 11 .
The highest RIs for red blood cells, hemoglobin, and hematocrit identified in men, compared to women, and higher RIs for platelets in women than in men, were also found in Ghana 3 , Oman 24 , and Brazil 1 . In this study, as well as in an investigation carried out in Morocco 25 , higher RIs of MCH and MCHC were observed in men compared to women. Differences between genders for erythrocytes, hemoglobin, hematocrit, MCH, MCHC, platelets, and platelet volume 1 have been documented, which can be explained by the effect of menstruation and the increased demand for iron 3,23 , hormonal influences of androgen 1,3,8 and the way in which erythropoiesis and megakaryopoiesis are regulated in men and women 3 .
Our findings agree with studies from Brazil 1 , Canada 8 , Korea 23 , and Morocco 25 , in which neutrophil levels were lower in men than in women, possibly due to impacts related to sexual development on immunity, in which estrogen stimulates an immune response, and some androgen hormones, such as testosterone, suppress the response to infection, with neutrophil levels being highest in females during puberty and adulthood 8,23 .
Studies carried out in Brazil 1 , Canada 8 , and China 22 found differences in monocyte RIs according to gender 8,22 , being higher in men, in line with our results. It is known that men can have higher monocyte counts than women, however the physiological and clinical aspects still need to be elucidated 8 . Higher eosinophil RIs in men than in women were also found in studies in Korea 23 , Morocco 25 , China 22 , Ghana 3 , and Brazil 1 . However, research is needed to investigate the reasons for the biological fluctuations of these leukocyte parameters in specific populations from different locations according to gender 9 .
The subtle differences found in hematocrit RIs in women, which are higher after 40 years of age, compared to 18 to 39 years of age, are justified by the variability of this parameter, which is similar throughout life 8 . Slight increase in hematocrit with age was found in Chinese adults. This finding is consistent with lower levels before menopause and higher levels after this period 2 . Red blood cell values tended to decrease with age in males, as in Chinese adults 2 . The decrease may be due to the gradual loss of androgen 2 . The lowest RIs from 60 years of age onward in men may be related to nutritional deficiency, occult malignancy, and anemia 2 .
For the RIs of white blood cells, the results found are similar to those of other studies 23,24 , in which a decline was observed with increasing age 8 . The findings reaffirm that two divisions by age could be made for this parameter, as in the Brazilian study 1 , as there were no differences between the two age groups of 18 to 39 and 40 to 59 years. Higher white blood cell counts in young adults of both genders, when compared to lower counts in the aged, reflect the development of the adaptive and acquired immune response as the immune system is more exposed to pathogens and antigens in the environment 8,23 .
In this study, the slight increase in eosinophil UL with age in men may be related to chronic infections in aged people who were not excluded 2 . This finding was present in Chinese adults of both genders 2 . Differences in eosinophil values can be attributed to allergic and parasitic diseases in apparently healthy adults 2 .
Studies in Canada 8 and Brazil 1 also identified a slight increase in MCV and RDW throughout life, with small changes in early adulthood, remaining constant after this phase 8 . A study in China identified that aged men and women had higher RIs for RDW 23 . MCV and RDW provide a classification of erythrocytes based on size and distribution 4 4 , consistent with the findings of this study. RDW is useful in identifying iron deficiency, ꞵ-thalassemia trait, inflammatory processes, and chronic infection 4 . It is also a predictor of mortality, and its increase with age is related to organic changes in aging and chronic diseases in this phase 1 .
In this study, significant differences in MCH values with age in men, being higher in the aged, was identified in a previous study 1 . It is noteworthy that the RIs for all age groups in Brazilian men were lower than the international hematrimetric classification (27 to 33.7 pg) 2 . The literature shows regional variations for HCM; in Chinese, RIs did not differ according to age and gender 2 , while in Moroccan men there were differences 25 . Although our study found differences in men, the international classification covers the same RIs values for MCH according to gender and age for adults 2 . In this sense, further investigations are desirable to clarify the implications of these differences in clinical practice.
The lowest platelet count identified in aged Brazilian men is in line with a study in Canada 8 and was found in men over 40 years of age in Iran 9 . A possible justification is the gradual decline of thrombopoietin, a hormone that regulates the production of platelets in adults. The occurrence of thrombocytopenia is more common in men than in women, and more frequent in the aged 8 .
In this study, RIs was established for 16 hemogram parameters according to race/color. In a previous research, RIs of five parameters were determined, and the values found were close to those described 1 . Although discreet, the differences found according to race/color for RIs of hemoglobin, MCH, MCHC, white blood cells and mean platelet volume, neutrophils and eosinophils, support the need to establish RIs for Brazilians. Differences in the blood count RIs were found in populations from other countries [1][2][3]8,10,24 , reinforcing the importance of considering geographic and ethnic-racial influences, as they may be related to factors such as genetics, nutrition, socioeconomic, cultural, and lifestyle factors, exposure to allergens, infections and parasitic loads in different locations 3 .
Limitations, such as the possibility of including patients without a previous diagnosis and the failure to obtain samples with 120 individuals for some strata of race/color, must be considered. However, due to the representativeness of the sample, it is emphasized that this study is close to the reality of the health conditions of the Brazilian population. Furthermore, the literature documents that it is possible to establish a RIs of 95% using up to 39 samples 13 . As for the losses attributed to the procedures of exclusion and removal of outliers, this is a conservative bias in clinical practice, as it was possible to estimate the RIs of blood counts for Brazilian adults and predict that the values found were close to the previous national study 1 , taking into account the recommendations to test other methodological approaches 13 . The RIs found here confer reliability and allow the generalization of the findings in a relatively safe way. Differences found in some hemogram parameters in Brazilian adults according to gender, age, and race/color show that there is a need to establish RIs that are adequate for the population. The results show the ethnic-racial influences on the RIs and can support the identification and prevention of diseases, as well as future research to validate the RIs of the Brazilian population, contributing to better interpretation, diagnostic accuracy, and quality of care and treatment offered.